Fig. 1.

Melphalan and CTLA-4 blockade in a model of melanoma. (A) B16 cells were treated in vitro with melphalan or vehicle control at 50 μM. After 24 hours, the expression of MHC class I, MHC class II, and PD-L1 was assessed by flow cytometry. This is a representative figure from the experiment performed 3 times. Fold change was calculated based upon mean fluorescence intensity change from untreated to treated. (B) Melphalan synergizes with CTLA-4 blockade. Mice with palpable allografted B16 tumors were treated with single intratumoral dose of melphalan, alone or followed by CTLA-4 blockade, 100 μg IP, every 3 days for 4 doses. Control mice received intratumoral injection of vehicle control and IP injection of isotype antibody. Shown are pooled data of 3 separate experiments (3–5 mice per treatment group per experiment, four experiments). (C,D) Combination therapy enhances the inflammatory environment of the tumor. (C) CD4 effector and CD8⁺ cells per gram of tumor in B16 tumors from the four treatment groups, P < 0.05 for combination versus control treatment. (D) CD4 and FoxP3 expression on cells from tumors from the four treatment groups. Experiments were performed 3 times; shown is representation of one experiment. (E) IFNγR^−/− mice had an improved median survival for melphalan plus CTLA-4 blockade over control or melphalan treated mice (combination (n = 9), median survival 22 days, control (n = 5), median survival 10 days, melphalan (n = 6), 12 days, P < 0.05). However, no combination mice were cured of tumors, suggesting the importance of IFNγ on the host.